magnitude of observed effects in the negative control hairpins, whereall effects are nonspecific.For 137 of the 147 lincRNAs (93%), knockdown caused a signifi-cant impact on gene expression (Supplementary Table 3), with anaverage of 175 protein-coding transcripts affected (range: 20–936)(Fig. 1c, Supplementary Fig. 2 and Supplementary Table 4). Theseresultsweresimilartothoseobtaineduponknockdownofthe40well-studied ES cell regulatory proteins: 38 (95%) showed significanteffects on gene expression, with an average of 207 genes affected(range: 28 (for Dnmt3l) to 1,187 (for Oct4)) (Fig. 1c, Supplemen-tary Fig. 2 and Supplementary Table 4). Although some individuallincRNAshavebeenfoundtoleadprimarilytogenerepression
,wefind that knockdown of the lincRNAs studied here largely led tocomparable numbers of activated and repressed genes (Supplemen-tary Fig. 2 and Supplementary Table 4). To assess off-target effectsfurther, we also profiled the effects of the second-best validatedshRNA targeting 10 randomly selected lincRNA genes. In all cases,secondshRNAsagainstthesametargetproducedsignificantlysimilarexpression changes (see Methods and Supplementary Table 5). Theseresults indicate that the vast majority of lincRNAs have functionalconsequences on overall gene expression of comparable magnitude(in terms of number of affected genes and impact on levels) to theknown transcriptional regulators in ES cells.
lincRNAs affect gene expression in
Following the observation that a few lincRNAs act in
, somerecent papers have claimed that most lincRNAs act primarily in
.Wefoundnoevidencetosupportthislatternotion:knockdownof only 2 lincRNAs showed effects on a neighbouring gene, only 13showed effects within a window of 10 genes on either side, and only 8showedeffectsongeneswithin300kb;theseproportionsarenogreaterthan observed for protein-coding genes (Supplementary Fig. 3 andSupplementary Table 6). In short, lincRNAs seem to affect expressionlargely in
.Our results contrast with a recent study that concluded thatlincRNAs act in
, based on the observation that knockdown of 7out of 12 lincRNAs affected expression of a gene within 300kb
. Theexplanationseemstobethatthethresholdintheprevious studyfailedto account for multiple hypothesis testing within the local region.Accounting for this, the effects on neighbouring genes are no greaterthan expected by chance and are consistent with our observationshere (see Methods).Although some lincRNAs can regulate gene expression in
,determining the precise proportion of
regulators requires moredirectexperimentalapproaches.Wenotethatourresultsareconsistentwith observed correlations between lincRNAs and neighbouring genes
, which may represent shared upstream regulation
or localtranscriptional effects
. In addition, the lincRNAs studied hereshouldbedistinguishedfromtranscriptsthatareproducedatenhancersites
, the function of which has yet to be determined.
lincRNAs maintain the pluripotent state
We next sought to investigate whether lincRNAs have a role in regu-lating the ES cell state. Regulation of the ES cell state involves twocomponents: maintaining the pluripotency program and repressing differentiation programs
. To determine whether lincRNAs have aroleinthemaintenanceofthepluripotencyprogram,westudiedtheireffects on the expression of Nanog, a key transcription factor that isrequired to establish
and uniquely marks the pluripotent state
.We infected ES cells carrying a luciferase reporter gene expressedfrom the endogenous
with shRNAs targeting lincRNAs or protein-coding genes. We monitored loss of reporteractivity after 8days relative to 25 negative control hairpins acrossbiological replicates (see Methods). To ensure that the observedeffectswerenotsimplyduetoareductionincellviability,weexcludedshRNAs that caused a reduction in cell numbers (see Methods,Supplementary Fig. 4 and Supplementary Table 7). Altogether, weidentified 26 lincRNAs that had major effects on endogenousNanog levels with many at comparable levels to the knockdown of the known protein-coding regulators of pluripotency such as Oct4and Nanog (Fig. 2a and Supplementary Table 7). This establishes thatthese lincRNAs have a role in maintaining the pluripotent state.To validate further the role of these 26 lincRNAs in regulating thepluripotent state, we knocked down these lincRNAs in wild-type EScells and measured mRNA levels of pluripotency marker genes
after 8days. In allcasesweobservedasignificantreductionintheexpressionofmultiplepluripotency markers with
90% showing a significant decrease inboth
levels(SupplementaryFig.5andSupplementary Tables8and9).Tocontrolforoff-targeteffects,westudiedadditionalhairpins targeting these lincRNAs. For 15 lincRNAs we had an effec-tive second hairpin. In all 15 cases, the second hairpin producedcomparable reductions in
expression levels, showing that theobservations were not due to off-target effects (Fig. 2b and Sup-plementary Table 10). Notably,
90% of lincRNA knockdownsaffecting Nanog reporter levels led to loss of ES cell morphology (Fig. 2c and Supplementary Figs 6 and 7). Thus, inhibition of these26 lincRNAs lead to an increased exit from the pluripotent state.
lincRNAs repress lineage programs
To determine if lincRNAs act in repressing differentiation programswe compared the overall gene expression patterns resulting fromknockdown of the lincRNAs to published gene expression patternsresulting from induced differentiation of ES cells
and assessedsignificance using a permutation-derived FDR
(see Methods).These states include differentiation into endoderm, ectoderm,neuroectoderm,mesodermandtrophectodermlineages.Asapositivecontrol for our analytical method, we confirmed the expected resultsthat the expression pattern caused by Oct4 knockdown was strongly associated with the trophoectoderm lineage
and the pattern causedby Nanog knockdown was strongly associated with endoderm differ-entiation
D i f f e r e n t i a l l y e x p r e s s e d g e n e s
27 negative controlsKD
0 1 , 4 0 0 4 0 0 2 0 0 6 0 0 1 , 0 0 0 8 0 0 1 , 2 0 0
D e n s i t y
Differentially expressed genes
Select besthairpinIdentifyaffectedgenesKnock-downInfect withshRNAControlEmbryonicstem cellsControlhairpinslincRNAhairpinsNanog40%
Functional affects of lincRNAs. a
, A schematic of lincRNAperturbationexperiments. ES cellsareinfected with shRNAs, knockdown levelis computed, the best hairpin is selected andprofiled on expression arrays, anddifferential gene expression is computed relative to negative control hairpins.
, Example of a lincRNA knockdown. Top: genomic locus containing thelincRNA. Bottom: heat-map of the 95 genes affected by knockdown of thelincRNA,expressionforcontrolhairpins(redline)andexpressionforlincRNAhairpins(blueline)areshown.
,Distributionofnumberofaffectedgenesuponknockdown of 147 lincRNAs (blue) and 40 well-known ES cell regulatory proteins (red). Points corresponding to five specific ES cell regulatory proteinsare marked.
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